Morphologically indistinguishable members of the Anopheles gambiae complex have remarkably distinct ecological adaptations, geographical distributions, and behaviors. This complex includes the major malaria vectors (A. arabiensis, A. gambiae), as well as minor vectors (A. bwambae, A. melas, A. merus) and nonvectors (A. quadriannulatus A, A. quadriannulatus B). Development of novel approaches to malaria management implies finding the ways to interrupt transmission by replacing populations of vectors with nonvectors. Our current knowledge about differences in genetic make-up among vectors and nonvectors is insufficient for implementing the successful population replacement. Inferring ancestral genomic features in the A. gambiae complex is crucial in identifying the evolutionary changes associated with the origin and loss of human blood choice, ecological and behavioral adaptations, and association with human habitats. These genomic changes should be targeted and manipulated so that malaria transmission can be reduced and eventually eliminated. Despite the availability of molecular markers and the genome sequence for A. gambiae, the phylogenetic relationships among the members are not fully resolved. Because of the recent origin of the complex, the high level of sequence similarity, genetic introgression, and shared molecular ancestral polymorphisms confound the ability to determine the direction of evolution unambiguously. The major goal of this exploratory R21 project is to determine the ancestral and derived chromosomal arrangements in the A. gambiae complex. The advantage of our approach is that it is based on observations that the ancestral arrangements are shared between ingroup and outgroup species and that the breakpoint structure can indicate the direction of chromosomal evolution. We plan to (1) identify chromosomal arrangements shared between the A. gambiae complex and the outgroup species A. stephensi and (2) determine derived structural features of inversion breakpoints such as the presence of transposable/repetitive elements and gene duplications. The comparative analysis of the ingroup and outgroup breakpoints will for the first time assess the uniqueness of the inversions and unambiguously determine the ancestral X and 2R arrangements in the complex. Although identifying and sequencing breakpoints are technically challenging tasks, the successful characterization of the 2La breakpoints and our preliminary studies suggest that our research aims are achievable. Our proposal has two specific aims: 1. To physically map the breakpoints of fixed inversions Xag, Xbcd, 2Rop to the chromosomes of the A. gambiae complex and A. stephensi and to calculate the inversion distances between ingroup and outgroup species. 2. To sequence and characterize the inversion breakpoints of Xc in A. arabiensis, Xa+, Xc+ in A. quadriannulatus, and 2Ro in A. merus, and the homologous sequences in A. stephensi. PUBLIC HEALTH RELEVANCE: This proposal will infer ancestral chromosome arrangements in the Anopheles gambiae complex. They are crucial in identifying the evolutionary genomic changes associated with the origin and loss of human blood choice, ecological and behavioral adaptations, and association with human habitats. These genomic changes should be targeted and manipulated so that malaria transmission can be reduced and eventually eliminated.